MBE Advance Access published June 19, 2014

Molecular Phylogeny, Biogeography, and Habitat Preference

Evolution of Marsupials
Kieren J. Mitchell,*,1 Renae C. Pratt,2 Laura N. Watson,3 Gillian C. Gibb,4 Bastien Llamas,1 Marta Kasper,1
Janette Edson,1 Blair Hopwood,1 Dean Male,1 Kyle N. Armstrong,1 Matthias Meyer,5 Michael Hofreiter,6
Jeremy Austin,1 Stephen C. Donnellan,1,7 Michael S. Y. Lee,1,7 Matthew J. Phillips,8 and Alan Cooper*,1
1
Australian Centre for Ancient DNA, School of Earth and Environmental Sciences, University of Adelaide, Adelaide, SA, Australia
2
Department of Evolution, Ecology and Genetics, The Australian National University, Canberra, ACT, Australia
3
Department of Obstetrics and Gynaecology, Research Centre for Reproductive Health, University of Adelaide, Adelaide, SA,
Australia
4
Institute of Agriculture and Environment, Massey University, Palmerston North, New Zealand
5
Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
6
Department of Biology, University of York, York, United Kingdom
7
South Australian Museum, Adelaide, SA, Australia
8
School of Earth, Environmental and Biological Sciences, Queensland University of Technology, Brisbane, QLD, Australia
*Corresponding author: E-mail: kieren.mitchell@adelaide.edu.au; alan.cooper@adelaide.edu.au.
Associate editor: Emma Teeling

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Abstract
Marsupials exhibit great diversity in ecology and morphology. However, compared with their sister group, the placental
mammals, our understanding of many aspects of marsupial evolution remains limited. We use 101 mitochondrial
genomes and data from 26 nuclear loci to reconstruct a dated phylogeny including 97% of extant genera and 58% of
modern marsupial species. This tree allows us to analyze the evolution of habitat preference and geographic distributions
of marsupial species through time. We found a pattern of mesic-adapted lineages evolving to use more arid and open
habitats, which is broadly consistent with regional climate and environmental change. However, contrary to the general
trend, several lineages subsequently appear to have reverted from drier to more mesic habitats. Biogeographic recon-
structions suggest that current views on the connectivity between Australia and New Guinea/Wallacea during the
Miocene and Pliocene need to be revised. The antiquity of several endemic New Guinean clades strongly suggests a
substantially older period of connection stretching back to the Middle Miocene and implies that New Guinea was
colonized by multiple clades almost immediately after its principal formation.
Key words: supermatrix, ancestral state reconstruction, mammal, mitochondrion.

Introduction
Modern marsupials originated in the Late Cretaceous and
today include over 300 known species distributed across
the Americas and Australasia (Wilson and Reeder 2005).
These species vary from gliding possums to hopping kanga-
roos and include obligate carnivores, specialized insectivores,
omnivores, and herbivores, and both diurnal and nocturnal
forms. They also occupy diverse habitats ranging from the wet
tropical rainforests of New Guinea and the Amazon to the
arid Australian interior. Marsupials occupy virtually every ter-
restrial, nonvolant niche occupied by placental analogs on
other continents. However, despite the great diversity of ad-
aptations displayed by living marsupials, studies attempting
to explain the drivers and constraints behind their evolution
have been limited compared with their sister group. This is
partly due to extensive gaps in the marsupial fossil record,
which obscure several critical periods in their evolutionary
history (Archer et al. 1999).
Article
Where the fossil record is depauperate, molecular phylog-
enies can be particularly useful tools for reconstructing the
tempo and mode of evolution. Time-calibrated phylogenies
are increasingly used to reconstruct biogeographic history
and test dispersal/vicariance hypotheses (Avise et al. 1987;
Avise 2000). Such phylogenies can also be used to model
the evolution of morphological and ecological traits in a phy-
logenetic context (Felsenstein 1985; Huey 1987; Pagel 1997).
Researchers have even attempted to use phylogenies of living
taxa to infer changes in the rate of speciation and extinction
through time (Alfaro et al. 2009; Stadler 2011; Hugall and
Stuart-Fox 2012). To fully investigate these issues and under-
stand the origin of extant marsupial diversity, it is necessary to
develop a comprehensive phylogeny as a template for explor-
ing marsupial macroevolution.
Since the turn of the century, great progress has been
made in resolving the relationships among marsupial taxa.
Eomarsupialia (Archer 1984; Phillips et al. 2006; Beck 2008;

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Mol. Biol. Evol. doi:10.1093/molbev/msu176 Advance Access publication May 30, 2014 1
Mitchell et al. . doi:10.1093/molbev/msu176
Meredith et al. 2011) has been identified as a clade comprising
the four Australasian orders: Diprotodontia (the kangaroos,
possums, wombats, and koala), Peramelemorphia (bandi-
coots and bilbies), Dasyuromorphia (including the
Tasmanian devil and the thylacine), and Notoryctemorphia
(marsupial moles). Eomarsupialia is nested within a paraphy-
letic collection of three endemic American lineages (Beck
2008; Meredith, Westerman, Case, et al. 2008; Meredith
et al. 2011): Didelphimorphia (opossums), Paucituberculata
(shrew-opossums), and Microbiotheria (the monito del
monte). The South American monito del monte
(Dromiciops gliroides) is particularly interesting as its close
affinity with the Australasian orders (together forming
Australidelphia) has implications for understanding the bio-
geography and dispersal of ancestral marsupials in the Late
Cretaceous and Paleocene (Beck 2012).
Unfortunately, the majority of past molecular studies have
only been able to test a limited range of hypotheses. Studies
seeking to resolve relationships among marsupial lineages
using nucleotide sequence data have either included dispa-
rate exemplar higher taxa but relatively few individual species
(Phillips et al. 2006; Beck 2008; Meredith, Westerman, Case,
et al. 2008; Meredith et al. 2011; Phillips and Pratt 2008;
Nilsson et al. 2010) or comprehensive species sampling but
only within restricted subclades (Meredith et al. 2008a, 2008b,
2010; Voss and Jansa 2009; Westerman et al. 2012). Often
these individual data sets have involved different genetic
loci, making their results difficult to combine when using
supermatrix approaches. To circumvent this problem, some
authors have turned to supertree methods (Cardillo et al.
2004; Bininda-Emonds et al. 2007). However, this approach
has several disadvantages including difficulties in quantifying
statistical support for inferred topologies (de Queiroz and
Gatesy 2007; von Haeseler 2012). As a result, phylogenies
focusing on marsupials have generally been rather poorly
resolved and inadequately dated, at least compared with pla-
cental mammals.
We have constructed a comprehensive phylogeny of
modern marsupials to better understand their evolution,
biogeography, and ecological history. We sequenced 69 mi-
tochondrial genomes using next-generation sequencing
technology and synthesized this with existing nuclear and
mitochondrial genomic nucleotide data to generate a super-
matrix that includes 97% of extant genera and 58% of modern
marsupial species.
Results and Discussion
Maximum likelihood (ML), Bayesian, and parsimony analyses
of the supermatrix resulted in well-resolved and concordant
phylogenetic trees (fig. 1 and supplementary fig. S1 and data
set S1, Supplementary Material online) with 85% of
nodes in the ML tree supported by ML bootstrap support
(MLBS) values  70% (fig. 1 and Supplementary fig. S1,
Supplementary Material online). Relationships among marsu-
pial orders and families are consistent with most recent mo-
lecular studies and generally receive high statistical support
(MLBS  97%). There is currently uncertainty surrounding
the precise affinities of two major clades: Macropodiformes
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(the kangaroos and wallabies) and Notoryctemorphia (the
marsupial moles). We robustly recover Macropodiformes
and Petauroidea (petauroid possums) as sister taxa
(MLBS = 85%), concordant with a recent study of major
mammal lineages (Meredith et al. 2011); this placement is
relatively novel, as several previous studies of mitochondrial
(Phillips and Pratt 2008) and nuclear loci (Meredith,
Westerman, Case, et al. 2008; Meredith et al. 2009), as well
as some aspects of morphology (Szalay 1994), favor an alter-
native grouping of Macropodiformes with Phalangeroidea
(phalangeroid possums). Additionally, our results suggest
that Notoryctemorphia is sister to a clade consisting of
Peramelemorphia and Dasyuromorphia, although support
for this grouping is moderate (MLBS = 68%). Relationships
below the family level are largely concordant with previous
estimates (e.g., Raterman et al. 2006; Beck 2008; Meredith et al.
2008b, 2009, 2010; Phillips and Pratt 2008; Westerman et al.
2008; Voss and Jansa 2009; Westerman et al. 2012).
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To estimate divergence times among marsupials, we em-
ployed a set of 14 established fossil-based node age con-
straints (supplementary table S1, Supplementary Material
online) (see Meredith, Westerman, Case, et al. 2008;
Meredith et al. 2008a, 2008b, 2009, 2010, 2011; Westerman
et al. 2012) for which taxonomic assignment is well justified.
These constraints range in age from a Late Cretaceous (71.2
Ma) maximum bound for the divergence between
Eomarsupialia and Microbiotheria, to a Middle Pliocene
(3.62 Ma) minimum bound for the divergence between the
bandicoot genera Isoodon and Perameles. Divergence dates
inferred in this study are broadly consistent with past esti-
mates based on molecular data (Beck 2008; Meredith,
Westerman, Case, et al. 2008, Meredith et al. 2008a, 2009,
2010, 2011; Westerman et al. 2012). The mean age of the
marsupial crown group is 86.8 Ma (95% highest posterior
density [HPD]: 79.7–94.6), whereas the four Australasian
orders diverge within a relatively brief window closely associ-
ated with the KPg boundary: 67–64 Ma. The divergence be-
tween the Australasian stem lineage and Microbiotheria only
slightly predates this period (~69 Ma) but is in turn preceded
by an approximately 14 Ma internode. Such a tight clustering
of divergences relatively deep in the tree is unlikely under a
standard birth–death diversification model. This may indicate
rapid diversification of ancestral australidelphians catalyzed
by ecological opportunity following the KPg mass extinction
or colonization of Antarctica/Australia from South America.
The latter hypothesis implies that the current distribution of
the monito del monte may reflect a back migration from
Antarctica/Australia to South America in the early
Cenozoic (Beck 2012).
Several previous studies have constructed comprehensive
marsupial phylogenies with similar aims to this study (Cardillo
et al. 2004; Bininda-Emonds et al. 2007). However, these used
supertree approaches and do not recover several widely ac-
cepted clades that are well supported by our analyses (e.g.,
Potoroidae, Echymiperinae, and Eomarsupialia). Similarly,
many marsupial genera in these previous studies are repre-
sented by polytomies, with resolution between congeneric
species completely lacking. Lack of resolution makes the
Marsupials . doi:10.1093/molbev/msu176 MBE
Caenolestidae
Paucituberculata
Didelphimorphia

Didelphidae

Microbiotheria Microbiotheriidae
Notoryctidae
Notoryctemorphia
Thylacomyidae
Peramelemorphia
Peramelidae
1
Thylacinidae
Myrmecobiidae
Australidelphia Dasyuromorphia
2

Dasyuridae
Eomarsupialia

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Phascolarctidae
Vombatidae
Burramyidae
Phalangeridae
Diprotodontia
3
Acrobatidae
Tarsipidae
Petauridae

Pseudocheiridae

Hypsiprymnodontidae
Potoroidae

Macropodidae

Late Cretaceous Paleocene Eocene Oligocene Miocene Pi Pt

80 70 60 50 40 30 20 10 0
Time (Ma)

FIG. 1. Time-calibrated phylogeny of 193 marsupial species based on ML topology (supplementary data set S1, Supplementary Material online). All
nodes are strongly supported by all three methods (ML bootstrap 90%, Bayesian posterior probability 0.95, and parsimony bootstrap 90%) unless
indicated by a dot. Black dot indicates a node appears in trees inferred using all three methods but is not strongly supported in at least one tree. Blue dot
indicates node absent in Bayesian tree, green dot indicates node absent in parsimony tree, and red dot indicates node absent in both Bayesian and
parsimony trees. Shading behind branches reflects ancestral distribution as estimated using Lagrange: light blue for the Americas; orange for Australia;
and red for New Guinea and Wallacea. Lineages that are inferred by Lagrange to be codistributed between Australia and New Guinea/Wallacea are
shaded purple. Numbers indicate New Guinean/Wallacean “old endemic” clades (see main text): 1) New Guinean/Wallacean bandicoots
(Echymiperinae and Peroryctinae), 2) “Murexia” dasyures, and 3) cuscuses (phalangerin possums). Geological epochs are marked on the x-axis: Late
Cretaceous, Paleocene, Eocene, Oligocene, Miocene, Pliocene (Pi), and Pleistocene (Pt). The Holocene (~11 ka to the present day) is not visible at this
scale. Parsimony optimization of distribution based on the ML topology gives comparable overall results (supplementary data set S1, Supplementary
Material online). Individual species names and locus coverage are presented in supplementary figure S1, Supplementary Material online.

dating analyses of Bininda-Emonds et al. (2007) inaccurate for
many clades, which is exacerbated by the fact that approxi-
mately 25% of node ages in their marsupial phylogeny were
interpolated rather than empirically estimated. Perhaps as a
result, mean node ages inferred by Bininda-Emonds et al.
(2007) are substantially older for many clades (e.g.,
Didelphimorphia) than those inferred by more recent studies
(e.g., Jansa et al. 2014), including our own. Consequently, the
direct dating and high resolution of our phylogeny make it a
more robust scaffold for testing macroevolutionary hypothe-
ses than existing supertrees.
Extant marsupials inhabit a diversity of habitat types rang-
ing from rainforest to desert. Figure 2 illustrates that habitat
preference is highly phylogenetically conserved. Using
Bayesian ancestral state reconstruction, we calculated the
probability that each node on the phylogeny (representing

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Mitchell et al. . doi:10.1093/molbev/msu176
Myrmecobiidae (numbat)
Thylacinidae (thylacine)
Peramelidae
(bandicoots)
Thylacomyidae (bilby)
Notoryctidae (marsupial moles)
Microbiotheriidae
(monito del monte)
Didelphidae
(opossums)
Wet-closed
Mesic-open
10
Arid
Caenolestidae
(shrew opossums)
FIG. 2. Ancestral habitat preference reconstruction using Bayesian optimization methods on the ML topology (fig. 1 and supplementary fig. S1 and data
set S1, Supplementary Material online). Boxes at tips reflect the modern state, whereas pie charts reflect the inferred probability of a particular state at
each ancestral node. Modern taxa inhabiting multiple habitat types are represented by an appropriate color gradient. Parsimony optimization of habitat
states gives comparable results (supplementary data set S1, Supplementary Material online).
an ancestral species) was associated with a given habitat
type: wet-closed (e.g., rainforest), mesic-open (e.g., sclerophyll
or grassland), or arid. A large proportion of the posterior
probability for the most basal nodes in the phylogeny was
associated with wet-closed environments (fig. 2). This sug-
gests that most of the primary divergences among extant
marsupial groups occurred in a rainforest context, consistent
with many paleontological studies (Travouillon et al. 2009).
However, the ancestral habitat on nodes at the base of the
australidelphian radiation is unclear, with intermediate
(mesic-open) habitat often having the highest probability.
Our results also suggest that adaptation to open and arid
habitats has evolved independently numerous times among
marsupials and that these events occurred relatively recently.
Among South American marsupials, transitions from wet-
closed to drier and more open environments appear unidi-
rectional and begin no earlier than the Miocene (Jansa et al.
2014) (fig. 2). The timing of these transitions follows the
Oligocene retraction of rainforest and expansion of wooded
savanna and grassland in southern South America (Ortiz-
Jaureguizar and Cladera 2006; Barreda and Palazzesi 2007;
Iglesias et al. 2011). Concordantly, extant open-habitat-
adapted opossum lineages (e.g., Thylamys and Lestodelphys)
are distributed primarily in southern Brazil, Paraguay,
Uruguay, Chile, and Argentina, whereas their respective
sister taxa are generally distributed in rainforests to the
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Dasyuridae (inc. quolls and Tasmanian devil)
Phascolarctidae (koala)
Vombatidae (wombats)
Burramyidae (pygmy possums)
Phalangeridae
(cuscuses and brushtail possums)
Acrobatidae
(inc. feathertail glider)
Tarsipidae
(honey possum)
Petauridae
80 (inc. wrist-winged
gliders)
70
60
50 Pseudocheiridae
(inc. ringtail possums)
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Time (Ma)
40
Hypsiprymnodontidae
30 (musky rat-kangaroo)
20 Potoroidae
(inc. potoroos and bettongs)
Macropodidae
(inc. kangaroos and wallabies)
north. In Australia, rainforest was the dominant habitat
type across most of the continent during the early
Cenozoic (Frakes et al. 1987): Open sclerophyll and grassland
did not become widespread until after the Middle Miocene,
and true arid zones did not develop until the Pliocene (Martin
2006; White 2006). Given this sequence of events, it is gener-
ally thought that the modern Australian arid biota evolved
from ancestral mesic forms and that the ancestral state for
these clades should consequently optimize as wet-closed
(Byrne et al. 2008, 2011). However, departures from this ex-
pected direction of habitat evolution are observed for several
Australasian taxa. For example, several clades and individual
species appear to have undergone reversions from mesic-
open to wet-closed habitats (fig. 2), particularly within
Macropodidae and Dasyuridae. Additionally, several groups
including Dasyuromorphia, Vombatiformes, and Trichosurini
appear to have made the transition from wet-closed to mesic-
open environments prior to the onset of widespread Miocene
aridification. One explanation for this observation is that
these older inferred transitions reflect lineages that survived
from drier periods in the Oligocene when limited open forest
habitats may have been available (Travouillon et al. 2009).
A limitation of molecular analyses is that they are based
purely on modern species. This may lead to inference of ar-
tificially old wet-closed to mesic-open transitions through
failure to sample extinct rainforest-adapted lineages. For
Marsupials . doi:10.1093/molbev/msu176
instance, support for an early origin of mesic-open habitats in
dasyuromorphs decreases when their close relatives, the arid-
adapted marsupial moles (Notoryctemorphia), are omitted
from the analysis. The two extant marsupial mole species are
associated exclusively with arid habitats, but extinct mesic
notoryctids are known from Miocene Riversleigh layers
(Archer et al. 2010). Additionally, extinct dasyurid and vom-
batiform taxa are known from Pleistocene rainforest assem-
blages (Cramb et al. 2009; Black et al. 2012). However, even if
we were to assume that no single wet-closed to mesic-open
transition is truly older than the Middle Miocene, these tran-
sitions are still temporally dispersed over a long period. This
suggests that evolution toward mesic-open and arid habitats
has been a gradual process rather than a concerted shift
across many Australasian clades in association with some
abrupt climatic event. This observation, along with evidence
for reversions from mesic-open to wet-closed habitats, indi-
cates a high level of ongoing plasticity among marsupials with
regard to habitat adaptation.
A large proportion of Australasian wet-closed forest line-
ages inhabit New Guinea and Wallacea rather than Australia
(Flannery 1995). The colonization timeline of these islands is
uncertain; long-distance overwater dispersal is improbable for
most marsupial taxa, and due to the geological complexity of
the region, evidence for the presence and duration of land
connections is equivocal (Metcalfe et al. 2001). Previous stud-
ies have attempted to use the age and distribution of marsu-
pial lineages to infer periods of biotic connection between
Australia and New Guinea/Wallacea (Aplin et al. 1993). We
implemented a similar approach on our phylogeny, using
biogeographic reconstructions to identify the temporal
origin of New Guinean/Wallacean marsupial taxa (fig. 1).
For taxa inferred to have colonized New Guinea/Wallacea
from Australia, the oldest probable dispersal time is repre-
sented by the divergence between the ancestor of a New
Guinean/Wallacean clade and its Australian sister taxon,
whereas the most recent probable dispersal time is marked
by the age of the New Guinean/Wallacean crown clade.
Inferred dispersal dates for any individual clade may be arti-
ficially young or old due to extinction of Australian lineages or
parallel colonization events, respectively. Thus, the power of
our study for testing biogeographical hypotheses comes from
the sheer number of independent dispersal events encom-
passed by our data set.
A vicariant origin of the New Guinean/Wallacean marsu-
pial fauna has previously been advanced based on early geo-
logical reconstructions (Flannery 1988). Under this scenario, a
substantial part of New Guinea was emergent and connected
to Australia until the Oligocene when the intervening Papuan
Basin was inundated (Dow 1976), thereby severing land con-
nection and preventing dispersal. Our inferred dates are uni-
formly too young to be consistent with this hypothesis.
Indeed, more recent geological reconstructions indicate that
in fact no substantial part of New Guinea was emergent until
the Miocene (van Ufford and Cloos 2005). Subsequently, it
has been suggested that the ancestors of New Guinean/
Wallacean marsupials dispersed no earlier than the latest
Miocene (Westerman et al. 2012) when a substantial drop
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in global sea levels may have permitted overland dispersal
(Hodell et al. 1986). Most of our inferred dispersal dates can
be reconciled with this scenario and/or more recent periods
of low sea level in the Pliocene and Pleistocene. However, the
age of several clades is unexpectedly old given this hypothesis.
The crown ages of the cuscuses (phalangerin possums),
New Guinean bandicoots (Peroryctinae and Echymiperinae),
and “Murexia” dasyures suggest a period of accessibility be-
tween Australia and New Guinea/Wallacea at the beginning
of the Late Miocene (fig. 1). The youngest inferred date for the
presence of cucuses in New Guinea/Wallacea is 10.65 Ma
(95% HPD minimum bound of the crown group); the analo-
gous values for New Guinean bandicoots and dasyures are
9.84 and 9.28 Ma, respectively. Although inference of such
early colonization dates could also be explained by multiple
parallel dispersals within each clade at the end of the Mio-
cene, the tight concordance among the crown ages of these
three clades strongly suggests a dispersal window 11–9 Ma.
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Similarly, sweepstakes long-range overwater dispersal is un-
likely to have generated three relatively simultaneous events.
This suggests that dispersal has been at least intermittently
possible between mainland Australia and New Guinea via
diffuse connection since the Middle Miocene. A period of
connection 11–9 Ma is broadly consistent with the origin
of the bulk of modern New Guinea, which formed as a
result of orogeny approximately 12 Ma (van Ufford and
Cloos 2005), and is consistent with the suggestion of a similar
period of dispersal based on microcomplement fixation
(Aplin et al. 1993).
This study presents a comprehensive molecular phylogeny
of marsupials and highlights substantial macroevolutionary
heterogeneity. The widely hypothesized trend of ancestral
wet-closed forest lineages evolving into open and arid
forms since the Middle Miocene appears to be overly simplis-
tic: Several lineages have evolved in the opposite direction.
Additionally, the taxonomic breadth of our phylogeny allows
us to compare the timing of numerous phylogenetically
independent dispersals from Australia to New Guinea/
Wallacea. The age and concordance of these events provides
substantial evidence of biological connectivity between New
Guinea and Australia coincident with New Guinea’s principal
formation approximately 12 Ma. Ultimately, our results sug-
gest that marsupial evolution in the Cenozoic was dynamic
and characterized by on-going ecological plasticity and op-
portunistic dispersal.
Materials and Methods
Data Set
We sequenced mitochondrial genomes (mitogenomes)
for 69 extant marsupial species (supplementary table S2,
Supplementary Material online). Sixty-three mitogenomes
were sequenced according to a previously published protocol
(Lerner et al. 2011) using a combination of multiplexed 454
pyrosequencing (Meyer et al. 2008) and traditional capillary
electrophoresis. First, we used long-range polymerase chain
reaction (see supplementary table S3, Supplementary
Material online, for primer sequences) to amplify each
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Mitchell et al. . doi:10.1093/molbev/msu176
mitogenome in several fragments. Each fragment was then
sheared, barcoded, and sequenced on a Roche/454 GS FLX.
Resulting sequencing reads were de novo assembled using a
previously published pipeline (Lerner et al. 2011): Sequences
were demultiplexed using “untag” (http://bioinf.eva.mpg.de/
pts/, last accessed June 5, 2014), and consensus sequences
were created using “newbler” (v. 2.0.00.20), SeqMan Pro
(Lasergene suite 8, DNASTAR), and MIA (http://sourceforge.
net/projects/mia-assembler/, last accessed June 5, 2014).
Remaining gaps and areas of low coverage were completed
using big dye terminator chemistry on an ABI 3130 (see sup-
plementary table S3, Supplementary Material online, for
primer sequences). The remaining six mitogenomes were ob-
tained via shotgun sequencing of raw extract on an Illumina
Genome Analyzer II (one lane per sample). Sequencing reads
were mapped to published mitogenomes of closely related
species using TMAP v3.2.2 (https://github.com/nh13/TMAP,
last accessed June 5, 2014) and samtools v1.4 (Li et al. 2009)
according to a previously published pipeline (Mitchell et al.
2014); duplicate reads were removed with PicardTools v1.79
(http://picard.sourceforge.net, last accessed June 5, 2014), and
a consensus sequence was generated in Geneious v6.1.2
(Biomatters; http://www.geneious.com, last accessed June 5,
2014).
New sequence data were combined with 32 previously
published mitogenomes and data from 26 nuclear loci to
form a single nucleotide supermatrix (supplementary fig. S1
and data set S2, Supplementary Material online). The final
data set included 193 marsupial species and ten outgroup
taxa: Two monotremes and eight placental mammals. The
supermatrix (43,616 bp) was 39% complete (excluding out-
groups), with the proportion of missing taxa per locus varying
from 12.5% to 88% (0–15% at the family level and 0% at the
order level). We divided this alignment into 72 discrete bins:
Codon positions of each protein-coding nuclear locus, first
and second codon positions of all H-strand mitochondrial
protein-coding genes (concatenated into one locus), individ-
ual introns, and stem and loop positions of mitochondrial
RNA-coding loci (all rRNAs and tRNAs were concatenated).
Mitochondrial third codon positions were discarded to min-
imize bias in branch length estimation arising from saturation.
These bins were analyzed using partitionfinder v0.9.2 (Lanfear
et al. 2012) to determine the most appropriate partitioning
scheme and substitution models (supplementary table S4,
Supplementary Material online) for downstream analysis in
RAxML v7.2.8, MrBayes v3.2.1, and MCMCtree (PAML v4.6).
Phylogenetics
Model-Based Methods
Tree topology was estimated under ML and Bayesian frame-
works using RAxML v7.2.8 (Stamatakis 2006) and MrBayes
v3.2.1 (Ronquist and Huelsenbeck 2003), respectively. Both
RAxML and MrBayes gave very similar trees (fig. 1 and sup-
plementary fig. S1 and data set S1, Supplementary Material
online). All analyses were repeated with and without out-
group taxa (placentals and monotremes) to assess the sensi-
tivity of the splits within marsupials to outgroup sampling;
6
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these analyses produced very similar trees (supplementary
data set S1, Supplementary Material online). Our RAxML
analysis comprised an ML search for the best-scoring tree
from 1,000 bootstrap replicates. MrBayes analyses were
undated (clock free), as dated (clock) analyses did not con-
verge. There were three runs: Each individual run employed
four Markov chains (one cold and three incrementally
heated) with default priors. Each chain ran for 107 genera-
tions, sampling every 500. Convergence in topology was as-
sessed using the average standard deviation of split
frequencies (<0.02), whereas convergence in individual pa-
rameter values was assessed through broadly overlapping dis-
tributions in Tracer v1.5 (http://tree.bio.ed.ac.uk/software/
tracer/, last accessed June 5, 2014) and effective sample
sizes more than 200. Trees generated by the three runs
were pooled before being summarized, and the first 25% of
trees from each run were discarded as burn-in.
Parsimony Methods
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Parsimony analyses used PAUP* (Swofford 2002), with most
parsimonious trees found via heuristic searches involving 100
random addition searches followed by a strict consensus;
bootstrapping was performed with 100 replicates of simple
heuristic searches (due to time constraints), followed by a
majority-rule consensus. All analyses were again repeated
with and without outgroup taxa, with similar results (supple-
mentary data set S1, Supplementary Material online).
Molecular Dating
We implemented 14 node calibrations across the marsupial
tree (supplementary table S1, Supplementary Material
online). Most constraints followed previous studies (e.g.,
Meredith, Westerman, Case, et al. 2008; Meredith et al.
2008a, 2008b, 2009, 2010, 2011; Westerman et al. 2012),
with minimum bounds being chosen based on the oldest
known occurrence of fossil crown-group taxa. Maximum
bounds were determined using either stratigraphic bounding
(Benton and Donoghue 2007) or phylogenetic bracketing
(Reisz and Müller 2004; Müller and Reisz 2005). To allow for
the patchiness of the marsupial fossil record, we followed the
conservative philosophy of Meredith, Westerman, Springer
(2008) when determining maxima: Stratigraphic bounding
maxima were judged based on absence of the lineage of in-
terest from two (rather than one) preceding fossil bearing
layers, whereas phylogenetic bracketing maxima were
judged based on the oldest known occurrence of at least
the second closest (rather than first closest) outgroup to
the target clade.
Molecular dating was performed using MCMCtree, within
the PAML v4.6 software package (Yang 2007), on the RAxML
tree topology. All calibrations (supplementary table S1,
Supplementary Material online) were implemented as uni-
form priors with hard minima and soft maxima (97.5%). To
maximize computational efficiency, we used the likelihood
approximation approach implemented in MCMCtree and
performed our analysis on ingroup taxa only. For the purposes
of our MCMCtree analyses, we defined individual time units
as 10 Ma, such that most node ages fell between 10 and 0.1
Marsupials . doi:10.1093/molbev/msu176
units. Among-partition rate variation was modeled using a
gamma distribution with the parameter values  = 1 and
 = 56. After a burn-in of 104 iterations, each chain was run
for 8  105 iterations sampling every 80 for a total of 104
samples. To ensure convergence and adequate sampling,
two separate runs were performed, and each parameter
was monitored in Tracer v1.5 (http://tree.bio.ed.ac.uk/soft
ware/tracer/, last accessed June 5, 2014). Parameter values
for one partition (out of 24) consistently failed to converge
due to a low number of informative sites; this partition was
consequently omitted from the final analysis (see supplemen-
tary table S4, Supplementary Material online).
Ancestral State Reconstruction
Bayesian trait reconstruction was performed on the final
dated phylogeny using the MCMC option for discrete state
evolution in BayesTraits v1.0 (Pagel et al. 2004). The analysis
was run for 106 iterations, sampling every 103. The first 10% of
samples were discarded as burn-in. To ensure convergence
and adequate sampling, each parameter was monitored in
Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/, last
accessed June 5, 2014). All modern taxa were coded as wet-
closed, mesic-open, or arid adapted (or a combination of
these) according to their distribution and habits (supplemen-
tary table S5, Supplementary Material online). An ordered
(wet-closed to mesic-open to arid) single-rate model was
used for evolution between the three character states. A
two-rate, ordered model allowing a separate rate for transi-
tions toward more arid environments and transitions toward
more mesic environments provided no significant improve-
ment in fit as determined by Bayes factor comparison
(
BF < 0.5); we thus used the simpler model in our analysis
to avoid overfitting. Both ordered models provided a substan-
tially better fit to the data than corresponding unordered
models (
BF > 5). To test the robustness of our results
under different optimization methods, we performed strict
parsimony state reconstructions (again using both ordered
and unordered models) in Mesquite (Maddison WP and
Maddison DR 2011) with concordant results (supplementary
data set S1, Supplementary Material online).
Geographical Range Evolution
We used Lagrange v20120508 (Ree and Smith 2008) to re-
construct ancestral distribution and infer dispersal times. The
model employed by this program attempts to account for
dispersal, fusion, and fission of geographic regions; speciation;
and local extinction. Distribution was coded as one (or a
combination) of three states (supplementary table S5,
Supplementary Material online): America, Australia, or New
Guinea/Wallacea. Individual species (including ancestral spe-
cies) were permitted to simultaneously inhabit the Americas
and Australia (to reflect potential connection scenarios in the
Paleocene), and Australia and New Guinea/Wallacea. The
model we employed allowed range shifts to occur bidirection-
ally but only between adjacent areas (Australia and New
Guinea/Wallacea, or America and Australia), because a
direct connection between the Americas and New Guinea/
MBE
Wallacea is not plausible. The most probable inherited range
for each node on the phylogeny is reflected in figure 1. To test
our results, we performed a strict parsimony state reconstruc-
tion in Mesquite (Maddison WP and Maddison DR 2011)
with concordant results (supplementary data set S1,
Supplementary Material online).
Supplementary Material
Supplementary tables S1–S5, figure S1, and data set S1 are
available at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/).
Acknowledgments
This study was funded by the Australian Research Council.
High-performance computing was provided by eResearch SA.
For access to tissue and DNA samples, the authors thank: J.
Patton and C. Cicero (Museum of Vertebrate Zoology); R.
Voss, S. Jansa, and E. Westwig (American Museum of
Downloaded from http://mbe.oxfordjournals.org/ by guest on August 24, 2016
Natural History); J. Cook (Museum of Southwestern
Biology); W. Foster (Adelaide Zoo); J. Graves (Australian
National University); South Australian Museum; and L.
Joseph and R. Palmer (Australian National Wildlife
Collection).
References
Alfaro ME, Santini F, Brock C, Alamillo H, Dornburg A, Rabosky DL,
Carnevale G, Harmon LJ. 2009. Nine exceptional radiations plus high
turnover explain species diversity in jawed vertebrates. Proc Natl
Acad Sci U S A. 106:13410–13414.
Aplin K, Baverstock PR, Donnellan SC. 1993. Albumin immunological
evidence for the time and mode of origin of the New Guinean
terrestrial mammal fauna. Sci New Guinea. 19:131–145.
Archer M. 1984. The Australian marsupial radiation. In: Archer M,
Clayton G, editors. Vertebrate zoogeography and evolution in
Australia. Carlisle (Australia): Hesperian Press. p. 633–708.
Archer M, Arena R, Bassarova M, Black K, Brammall J, Cooke B, Creaser
P, Crosby K, Gillespie A, Godthelp H, et al. 1999. The evolutionary
history and diversity of Australian mammals. Aust Mammal. 21:
1–45.
Archer M, Beck R, Gott M, Hand S, Godthelp H, Black K. 2010. Australia’s
first fossil marsupial mole (Notoryctemorphia) resolves controver-
sies about their evolution and palaeoenvironmental origins. Proc R
Soc B. 278:1498–1506.
Avise JC. 2000. Phylogeography: the history and formation of species.
Cambridge (MA): Harvard University Press.
Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, Neigel JE, Reeb CA,
Saunders NC. 1987. Intraspecific phylogeography—the mitochon-
drial–DNA bridge between population-genetics and systematics.
Annu Rev Ecol Syst. 18:489–522.
Barreda V, Palazzesi L. 2007. Patagonian vegetation turnovers during the
Paleogene–Early Neogene: origin of arid-adapted floras. Bot Rev. 73:
31–50.
Beck RM. 2012. An ‘ameridelphian’ marsupial from the early Eocene of
Australia supports a complex model of Southern Hemisphere mar-
supial biogeography. Naturwissenschaften 99:715–729.
Beck RMD. 2008. A dated phylogeny of marsupials using a molecular
supermatrix and multiple fossil constraints. J Mammal. 89:175–189.
Benton MJ, Donoghue PCJ. 2007. Paleontological evidence to date the
tree of life. Mol Biol Evol. 24:26–53.
Bininda-Emonds ORP, Cardillo M, Jones KE, MacPhee RDE, Beck RMD,
Grenyer R, Price SA, Vos RA, Gittleman JL, Purvis A. 2007. The
delayed rise of present-day mammals. Nature 446:507–512.
Black K, Archer M, Hand S, Godthelp H. 2012. The rise of Australian
marsupials: a synopsis of biostratigraphic, phylogenetic,
7
Mitchell et al. . doi:10.1093/molbev/msu176
palaeoecologic and palaeobiogeographic understanding. In: Talent J,
editor. Earth and life: Global biodiversity, extinction intervals and
biogeographic perturbations through time. Dordrecht (The
Netherlands): Springer. p. 983–1078.
Byrne M, Steane DA, Joseph L, Yeates DK, Jordan GJ, Crayn D, Aplin K,
Cantrill DJ, Cook LG, Crisp MD, et al. 2011. Decline of a biome:
evolution, contraction, fragmentation, extinction and invasion of
the Australian mesic zone biota. J Biogeogr. 38:1635–1656.
Byrne M, Yeates DK, Joseph L, Kearney M, Bowler J, Williams MAJ,
Cooper S, Donnellan SC, Keogh JS, Leys R, et al. 2008. Birth of a
biome: insights into the assembly and maintenance of the
Australian arid zone biota. Mol Ecol. 17:4398–4417.
Cardillo M, Bininda-Emonds ORP, Boakes E, Purvis A. 2004. A species-
level phylogenetic supertree of marsupials. J Zool (Lond). 264:11–31.
Cramb J, Hocknull S, Webb GE. 2009. High diversity Pleistocene rain-
forest dasyurid assemblages with implications for the radiation of
the Dasyuridae. Austral Ecol. 34:663–669.
de Queiroz A, Gatesy J. 2007. The supermatrix approach to systematics.
Trends Ecol Evol. 22:34–41.
Dow DB. 1976. A geological synthesis of Papua New Guinea. Vol. 201.
Canberra (Australia): Bureau of Mineral Resources, Geology and
Geophysics. p. 41.
Felsenstein J. 1985. Phylogenies and the comparative method. Am Nat.
125:1–15.
Flannery T. 1995. Mammals of New Guinea. Chatswood (Australia):
Reed Books.
Flannery TF. 1988. Origins of the Australo-Papuan land mammal fauna.
Aust Zool Rev. 1:15–24.
Frakes LA, McGowran B, Bowler JM. 1987. Evolution of Australian en-
vironments. In: Dyne GR, Walton DW, editors. Fauna of Australia.
Vol. 1A. Canberra (Australia): Australian Government Publishing
Service. p. 1–16.
Hodell DA, Elmstrom KM, Kennett JP. 1986. Latest Miocene benthic
d18O changes, global ice volume, sea-level and the Messinian salinity
crisis. Nature 320:411–414.
Huey RB. 1987. Phylogeny, history and the comparative method. In:
Feder ME, Bennett AF, Burgen W, Huey RB, editors. New directions
in ecological physiology. Cambridge (United Kingdom): Cambridge
University Press. p. 76–98.
Hugall AF, Stuart-Fox D. 2012. Accelerated speciation in colour-poly-
morphic birds. Nature 485:631–634.
Iglesias ARI, Artabe AE, Morel EM. 2011. The evolution of Patagonian
climate and vegetation from the Mesozoic to the present. Biol J Linn
Soc. 103:409–422.
Jansa SA, Barker FK, Voss RS. 2014. The early diversification of didelphid
marsupials: a window into South America’s “splendid isolation”.
Evolution 68:684–695.
Lanfear R, Calcott B, Ho SY, Guindon S. 2012. PartitionFinder: combined
selection of partitioning schemes and substitution. Mol Biol Evol. 29:
1537–1719.
Lerner HR, Meyer M, James HF, Hofreiter M, Fleischer RC. 2011.
Multilocus resolution of phylogeny and timescale in the extant
adaptive radiation of Hawaiian honeycreepers. Curr Biol. 21:
1838–1844.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G,
Abecasis G, Durbin R, Subgroup GPDP. 2009. The sequence align-
ment/map (SAM) format and SAMtools. Bioinformatics 25:
2078–2079.
Maddison WP, Maddison DR. 2011. Mesquite: a modular system for
evolutionary analysis. Version 2.75 [Internet]. [cited 2014 Jun 5].
Available from: http://mesquiteproject.org.
Martin HA. 2006. Cenozoic climatic change and the development of the
arid vegetation in Australia. J Arid Environ. 66:533–563.
Meredith RW, Janecka JE, Gatesy J, Ryder OA, Fisher CA, Teeling EC,
Goodbla A, Eizirik E, Simao TLL, Stadler T, et al. 2011. Impacts of the
Cretaceous terrestrial revolution and KPg extinction on mammal
diversification. Science 334:521–524.
Meredith RW, Mendoza MA, Roberts KK, Westerman M, Springer MS.
2010. A phylogeny and timescale for the evolution of
8
MBE
Pseudocheiridae (Marsupialia : Diprotodontia) in Australia and
New Guinea. J Mamm Evol. 17:75–99.
Meredith RW, Westerman M, Case JA, Springer MS. 2008. A phylogeny
and timescale for marsupial evolution based on sequences for five
nuclear genes. J Mamm Evol. 15:1–36.
Meredith RW, Westerman M, Springer MS. 2008a. A phylogeny and
timescale for the living genera of kangaroos and kin
(Macropodiformes : Marsupialia) based on nuclear DNA sequences.
Aust J Zool. 56:395–410.
Meredith RW, Westerman M, Springer MS. 2008b. A timescale and phy-
logeny for “Bandicoots” (Peramelemorphia : Marsupialia) based on
sequences for five nuclear genes. Mol Phylogenet Evol. 47:1–20.
Meredith RW, Westerman M, Springer MS. 2009. A phylogeny of
Diprotodontia (Marsupialia) based on sequences for five nuclear
genes. Mol Phylogenet Evol. 51:554–571.
Metcalfe I, Smith JMB, Morwood M, Davidson I. 2001. Faunal and floral
migrations and evolution in SE Asia-Australasia. Lisse (The
Netherlands): Swets & Zeitlinger Publishers.
Meyer M, Stenzel U, Hofreiter M. 2008. Parallel tagged sequencing on
the 454 platform. Nat Protoc. 3:267–278.
Mitchell KJ, Wood JR, Scofield RP, Llamas B, Cooper A. 2014. Ancient
mitochondrial genome reveals unsuspected taxonomic affinity of
Downloaded from http://mbe.oxfordjournals.org/ by guest on August 24, 2016
the extinct Chatham duck (Pachyanas chathamica) and resolves
divergence times for New Zealand and sub-Antarctic brown teals.
Mol Phylogenet Evol. 70:420–428.
Müller J, Reisz RR. 2005. Four well-constrained calibration points from
the vertebrate fossil record for molecular clock estimates. Bioessays
27:1069–1075.
Nilsson MA, Churakov G, Sommer M, Van Tran N, Zemann A, Brosius J,
Schmitz J. 2010. Tracking marsupial evolution using archaic genomic
retroposon insertions. PLoS Biol. 8:e1000436.
Ortiz-Jaureguizar E, Cladera GA. 2006. Paleoenvironmental evolution of
southern South America during the Cenozoic. J Arid Environ. 66:
498–532.
Pagel M. 1997. Inferring evolutionary processes from phylogenies. Zool
Scr. 26:331–348.
Pagel M, Meade A, Barker D. 2004. Bayesian estimation of ancestral
character states on phylogenies. Syst Biol. 53:673–684.
Phillips MJ, McLenachan PA, Down C, Gibb GC, Penny D. 2006.
Combined mitochondrial and nuclear DNA sequences resolve the
interrelations of the major Australasian marsupial radiations. Syst
Biol. 55:122–137.
Phillips MJ, Pratt RC. 2008. Family-level relationships among
the Australasian marsupial “herbivores” (Diprotodontia : Koala,
wombats, kangaroos and possums). Mol Phylogenet Evol. 46:
594–605.
Raterman D, Meredith RW, Ruedas LA, Springer MS. 2006. Phylogenetic
relationships of the cuscuses and brushtail possums (Marsupialia :
Phalangeridae) using the nuclear gene BRCA1. Aust J Zool. 54:
353–361.
Ree RH, Smith SA. 2008. Maximum likelihood inference of geographic
range evolution by dispersal, local extinction, and cladogenesis. Syst
Biol. 57:4–14.
Reisz RR, Müller J. 2004. Molecular timescales and the fossil record: a
paleontological perspective. Trends Genet. 20:237–241.
Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic
inference under mixed models. Bioinformatics 19:1572–1574.
Stadler T. 2011. Mammalian phylogeny reveals recent diversification rate
shifts. Proc Natl Acad Sci U S A. 108:6187–6192.
Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylo-
genetic analyses with thousands of taxa and mixed models.
Bioinformatics 22:2688–2690.
Swofford DL. 2002. PAUP*. Phylogenetic analysis using parsimony (*and
other methods). Sunderland (MA): Sinauer Associates.
Szalay FJ. 1994. Evolutionary history of the marsupials and an analysis of
osteological characters. Cambridge (MA): Cambridge University
Press.
Travouillon KJ, Legendre S, Archer M, Hand SJ. 2009. Palaeoecological
analyses of Riversleigh’s Oligo-Miocene sites: implications for Oligo-
Marsupials . doi:10.1093/molbev/msu176
Miocene climate change in Australia. Palaeogeogr Palaeoclimatol
Palaeoecol. 276:24–37.
van Ufford AQ, Cloos M. 2005. Cenozoic tectonics of New Guinea.
AAPG Bull. 89:119–140.
von Haeseler A. 2012. Do we still need supertrees? BMC Biol. 10:13.
Voss RS, Jansa SA. 2009. Phylogenetic relationships and classification of
didelphid marsupials, an extant radiation of new world metatherian
mammals. Bull Am Mus Nat Hist. 322:1–177.
Westerman M, Kear BP, Aplin K, Meredith RW, Emerling C, Springer MS.
2012. Phylogenetic relationships of living and recently extinct ban-
dicoots based on nuclear and mitochondrial DNA sequences. Mol
Phylogenet Evol. 62:97–108.
MBE
Westerman M, Young J, Krajewski C. 2008. Molecular relationships of
species of Pseudantechinus, Parantechinus and Dasykaluta
(Marsupialia: Dasyuridae). Aust Mammal. 29:201–212.
White ME. 2006. Environments of the geological past. In: Merrick JR,
Archer M, Hickey GM, Lee MSY, editors. Evolution and biogeogra-
phy of Australasian vertebrates. Sydney (Australia): Auscipub Pty
Ltd.
Wilson DE, Reeder DM. 2005. Mammal species of the world: a taxo-
nomic and geographic reference. Baltimore (MD): The Johns
Hopkins University Press.
Yang Z. 2007. PAML4: a program package for phylogenetic analysis by
maximum likelihood. Mol Biol Evol. 24:1586–1591.
Downloaded from http://mbe.oxfordjournals.org/ by guest on August 24, 2016